Protocol to identify human subcellular alternative protein interactions using cross-linking mass spectrometry

Abstract

Summary: Since the start of mass-spectrometry-based proteomics, proteins from non-referenced open reading frames or alternative proteins (AltProts) have been overlooked. Here, we present a protocol to identify human subcellular AltProt and decipher some interactions using cross-linking mass spectrometry. We describe steps for cell culture, in cellulo cross-link, subcellular extraction, and sequential digestion. We then detail both liquid chromatography-tandem mass spectrometry and cross-link data analyses. The implementation of a single workflow allows the non-targeted identification of signaling pathways involving AltProts.For complete details on the use and execution of this protocol, please refer to Garcia-del Rio et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Bioinformatics
• Proteomics
• Mass Spectrometry
• Systems Biology